PROJECT SUMMARY Dilated cardiomyopathy (DCM) is a severe and prevalent inherited cardiac defect, characterized by ventricular chamber enlargement and systolic dysfunction. Although DCM is commonly associated with mutations in genes associated with contractility and other myocyte-specific functions, fibrotic and endothelial dysfunctions in patients suggest non-myocytes can influence disease pathogenesis and progression. Cardiac myocytes actively secrete a diverse array of proteins and vesicles into the extracellular milieu, the contents of which can change dynamically in response to stress and disease, suggesting a potential avenue of crosstalk communicating disease status between myocytes and non-myocytes. Thus far, our understanding of the cardiac secretomes is incomplete, hampered by difficulty of differentiating proteins secreted by the heart vs. other organs in patient plasma. To overcome this challenge, we propose to leverage cutting-edge iPSC technology, genome-editing technology, and proteomics technology to discover and validate cardiac secretomes and the crosstalk signaling pathways they regulate in the context of DCM pathogenesis. To identify the complement of secreted proteins from healthy and diseased cardiac cells, we first propose to generate human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from DCM patients with three common sarcomeric mutations. To clarify the detailed molecular mechanisms, we will conduct structural, electrophysiological, developmental, transcriptomic, and mechanistic analyses using patient- specific as well as genome-edited isogenic iPSC-CMs. This isogenic human iPSC platform will then be used to systematically discover the (i) secreted proteins and (ii) secreted exosomes of cardiac cells using large- scale proteomics platforms capable of quantifying hundreds of low-abundance proteins of interest. To confirm the signaling modality of secreted proteins, we will perform detailed transcriptomic and functional analysis of iPSC-derived endothelial cells (iPSC-ECs) and iPSC-derived cardiac fibroblasts (iPSC-CFs) co-cultured with diseased vs. healthy iPSC-CMs using high-throughput platforms. We anticipate that the successful completion of these studies will lead to new mechanistic insights into DCM pathogenesis, and help identify novel therapeutic targets that can impede and revert disease crosstalk signaling between myocytes and non- myocytes in the diseased heart.